REACTION-KINETICS FOR NITROSATION OF CYSTEINE AND GLUTATHIONE IN AEROBIC NITRIC-OXIDE SOLUTIONS AT NEUTRAL PH - INSIGHTS INTO THE FATE AND PHYSIOLOGICAL-EFFECTS OF INTERMEDIATES GENERATED IN THE NO O-2 REACTION/

The critical regulatory function of nitric oxide (NO) in many physiolo gic processes is well established. However, in an aerobic aqueous envi ronment NO is known to generate one or more reactive and potentially t oxic nitrogen oxide (NOx) metabolites. This has led to the speculation that mechanisms must exist in vive by which these reactive intermedia tes are detoxified, although the nature of these mechanisms has yet to be elucidated. This report demonstrates that among the primary bioorg anic products of the reaction of cellular constituents with the interm ediates of the NO/O-2 reaction are S-nitrosothiol (S-NO) adducts. Anae robic solutions of NO are not capable of nitrosating cysteine or gluta thione, while S-NO adducts of these amino acids are readily formed in the presence of O-2 and NO. Investigation of the kinetics for the form ation of these S-NO adducts has revealed a rate equation of d[RSNO]/dt = k(SNO)[NO](2)[O-2], where k(SNO) = (6 +/- 2) X 10(6) M(-2) s(-1), a value identical to that for the formation of reactive intermediates i n the autoxidation of NO. Competition studies performed with a variety of amino acids, glutathione, and azide have shown that cysteine resid ues have an affinity for the NOx species that is 3 orders of magnitude greater than that of the nonsulfhydryl amino acids, and > 10(6) times greater than that of the exocyclic amino groups of DNA bases. The dip eptide alanyltyrosine reacts with the intermediates of the NO/O-2 reac tion with an affinity 150 times less than that of the sulfhydryl-conta ining compounds. Furthermore, Chinese hamster V79 lung fibroblasts dep leted of glutathione display enhanced cytotoxicity on exposure to NO. Together, these results suggest that the S-NO adduct of glutathione ma y represent a physiological scavenger of NOx species and that enzymes containing cysteine residues critical to their function may be subject to inhibition by reactive intermediates generated in the NO/O-2 react ion.